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A new ERK2 binding protein, Naf1, attenuates the EGF/ERK2 nuclear signaling
Shengliang Zhang1, Masaya Fukushi, Shinichi Hashimoto
1Department of Molecular Cellular Oncology, Graduate School, Tokyo Medical and Dental University, Tokyo 113, Japan.
Abstract:
Extracellular signal regulated kinase1/2 (ERK1/2), an important factor in signal transduction, controls cell growth, differentiation, and death. To elucidate the details of the mechanism of ERK1/2 signaling in human cells, we isolated Nef-associated factor 1 alpha (Naf1 alpha) by a yeast two-hybrid system, which bound to human ERK2. The binding was confirmed by a pull-down assay in vitro and immunoprecipitation in vivo. Upon EGF treatment, Naf1 alpha was phosphorylated by the EGF/MEK/ERK2 signal transduction pathway. To identify the role of Naf1 alpha in the ERK2 signaling, Naf1 alpha-expressing Saos-2 cells were analyzed for ERK2 nuclear translocation and activation of its downstream target. Overexpression of Naf1 alpha suppressed ERK2 entering into the nucleus and inhibited the ERK2-dependent Elk1-driven luciferase transcription, suggesting Naf1 alpha to be an attenuator of activated ERK2 signaling.
Insights
Nef-associated factor 1 alpha (Naf1 alpha) binds and is phosphorylated by ERK2 signaling. Overexpressing Naf1 alpha suppresses ERK2 nuclear translocation, attenuating downstream gene activation.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Signal transduction mechanisms
Background:
- Extracellular signal-regulated kinases (ERK1/2) are crucial regulators of cell growth, differentiation, and death.
- Understanding the precise mechanisms of ERK1/2 signaling in human cells is essential for comprehending cellular processes.
Purpose of the Study:
- To identify novel proteins interacting with ERK2.
- To elucidate the role of Naf1 alpha in the ERK2 signaling pathway.
Main Methods:
- Yeast two-hybrid system for protein interaction discovery.
- Pull-down assays and immunoprecipitation for binding validation.
- Analysis of ERK2 nuclear translocation and downstream transcriptional activity in Naf1 alpha-expressing cells.
Main Results:
- Naf1 alpha was identified as an ERK2-binding protein.
- Naf1 alpha is phosphorylated by the EGF/MEK/ERK2 pathway.
- Overexpression of Naf1 alpha inhibited ERK2 nuclear translocation and downstream Elk1-driven transcription.
Conclusions:
- Naf1 alpha acts as a negative regulator or attenuator of activated ERK2 signaling.
- Naf1 alpha influences ERK2's ability to translocate to the nucleus and activate target genes.